To understand the mechanism of action of any hormone in vitro techniques must be employed. For the case of the hypothalamic releasing hormones the use of the in vitro pituitary is not sufficient, primarily because the anterior pituitary synthesizes and secretes at least six different hormones. We have been able to develop a technique whereby we can obtain a highly enriched population of normal pituitary thyrotrophs. These cells are responsive to thyrotropin releasing hormone (TRH) and grow well in monolayer culture. With this system we plan to examine in detail two specific steps in the release of the anterior pituitary hormones: 1) the role played by calcium and 2) the electrical interaction between a releasing hormone and its receptor on the pituitary cell membrane. We have determined that calcium of intracellular origin is essential for pituitary hormone secretion. Further, such calcium, upon stimulation, becomes tightly bound. Our immediate objectives are to identify where in the cell such Ca ions is mobilized from, where it goes, what route it takes and what factors regulate its movement. For the electrical studies we are primarily concerned with determining the nature, if any, of the electrical interaction between TRH and its receptor, and assessing its importance in the release of TSH. Long range studies will begin to deal with the interaction of calcium and the cyclic AMP-protein kinase system as an intracellular regulator of pituitary hormone secretion.